PRMT6-mediated ADMA promotes p62 phase separation to form a negative feedback loop in ferroptosis

Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.

respectively into HEK293T cells.And co-IP was performed with anti-GFP, followed by immunoblotting with ADMA, GFP and Myc antibodies.D. HEK293T cells were co-transfected with Flag-p62 and siRNAs of arginine demethylases (siKDM3A, siKDM4A, siKDM4C, siKDM5C, siKDM6B or JMJD6) respectively.And IP was performed with anti-Flag, followed by immunoblotting with ADMA and Flag antibodies.E. HEK293T cells were co-transfected with Flag-p62 and siJMJD6s, IP was performed with anti-Flag, followed by immunoblotting with ADMA, JMJD6 and Flag antibodies.And relative quantified p62 ADMA level normalized to the immunoprecipitated Flag-p62 were shown.
F. GFP-p62 was co-transfected with Myc-PRMT6 and Flag-JMJD6 into HEK293T cells.And co-IP was performed with anti-GFP, followed by immunoblotting with ADMA, GFP and Myc antibodies.C.After p62 knockdown in MIAPACA-II or BxPC3 cells, cells were incubated with 3 μM RSL3 for 24 h, qPCR was adopted to measure the mRNA expression of NQO1.D-E.After MIAPACA-II or BxPC3 cells were transfected with PRMT6 siRNAs, cells were then incubated with 3 μM RSL3 (A) or 2 μM ML162 (B) for 24 h, western blotting was applied to detect the protein level of Nrf2, Keap1 and PRMT6.And relative quantified protein level of Nrf2 and Keap1 normalized to Actin were shown.
F. After PRMT6 knockdown in MIAPACA-II or BxPC3 cells, cells were incubated with RSL3 for 24 h, qPCR was adopted to measure the mRNA expression of NQO1.

Figure
Figure S2.p62 is important to Nrf2 upregulation during ferroptosis activation A-B.The protein level of Nrf2, Keap1 and p62 in Hela cells with p62 knockdown and 2 μM ML162 (A) or 8 μM Erastin (B) treatment for 24 h, were determined by western blotting.Actin was used as a loading control.And relative quantified protein level of Nrf2, Keap1 and LC3-II normalized to Actin were shown below the corresponding band.

Figure S3 .
Figure S3.Ferroptosis inducers promoted p62 phase separation to recruit Keap1 into p62 body A. p62 distribution in Hela cells cultured with 8 μM Erastin for 12 h, were fixed and treated with or without 5% 1,6-Hexanediol (1,6-HD), was determined by immunofluorescence (IF).And the numbers of p62 body per cell (n = 5) were counted and shown and mean ± SD.B. Soluble-insoluble fractions of p62 after RSL3 or ML162 24 h incubation in

9B.
HEK293T cells were co-transfected with Flag-p62 and Myc-PRMT6, then overnight treated with AdoX (5 μM) or MS049 (10 μM), and IP was performed with anti-Flag, followed by immunoblotting with ADMA, Myc and Flag antibodies.C. HEK293T cells were co-transfected with Flag-p62 or its C terminal truncations (deleted from C terminal as shown) and Myc-PRMT6, and co-IP was performed with anti-Flag, then immunoblotting with Myc and Flag antibodies.D. HEK293T cells were co-transfected with Flag-p62, its N terminal truncations (deleted from N terminal as shown) or LB domain deletion (△LB) mutant and Myc-PRMT6, and co-IP was performed with anti-Flag, then immunoblotting with Myc and Flag antibodies.E. HEK293T cells were co-transfected with Flag-p62 and Myc-PRMT6 or its deletions as shown, and co-IP was performed with anti-Myc, then immunoblotting with Myc and Flag antibodies.

Figure S7 .
Figure S7.Ferroptosis inducers increased the oligomerization of p62 dependent on R183 and R217 methylation A and B. Soluble-insoluble fractions of Flag-p62-WT (A) or Flag-p62-2RK (B) mutant after RSL3 or ML162 12 h incubation in Hela cells with Flag-p62-WT or Flag-p62-2RK transient transfection were determined by western blotting.And relative quantified protein level of Flag-p62 normalized to Actin in soluble and insoluble fractions were shown.

Figure S8 .
Figure S8.Activation of PRMT6-mediated p62 ADMA resulted in ferroptosis resistance A. PRMT6 knockdown Hela cells were incubated with 8 μM Erastin for 24 h, and western blotting was applied to detected the protein level of Nrf2, Keap1 and PRMT6, actin was used as a loading control.And relative quantified protein level of Nrf2 and Keap1 normalized to Actin were shown.B. PRMT6 knockdown Hela cells were incubated with Erastin for 24 h, qPCR was adopted to measure the mRNA expression of HMOX1.C. PRMT6 knockdown Hela cells were incubated with RSL3, ML162 or Erastin for 24 h, qPCR was performed to measure the mRNA expression of NQO1.D. PRMT6 knockdown Hela cells were incubated with Erastin for 48 h, together with or without Lip-1, cell viability was determined by MTS assay.E. p62-KO cells were transfected with PRMT6 siRNAs, then incubated with 1 μM ML162 for 48 h, and cell viability was determined by MTS assay.PRMT6 knockdown was validated by immunoblotting.

Figure S9 .
Figure S9.Ferroptosis inducers augmented PRMT6-dependent ADMA to promote p62 body formation in pancreatic cancer cells A. Protein level of p62 and PRMT6 were determined by western blotting in

Figure S10 .
Figure S10.Ferroptosis inducers activated p62-Keap1-Nrf2 axis in pancreatic cancer cells A-B.After MIAPACA-II or BxPC3 cells were transfected with p62 siRNAs, cells were then incubated with 3 μM RSL3 (A) or 2 μM ML162 (B) for 24 h, western blotting was applied to detect the protein level of Nrf2, Keap1 and p62, actin was used as a loading control.And relative quantified protein level of Nrf2 and Keap1 normalized to Actin were shown.

Figure S12 .
Figure S12.Stably knockdown of p62 or PRMT6 sensitized ferroptosis in vitro A-B.Knockdown of p62 (A) or PRMT6 (B) in MIAPACA-II stable knockdown cells (shp62 or shPRMT6) was validated by western blotting.C. Cell viability of parental MIAPACA-II cells or stable knockdown cells (shp62